Pax6 Directly Down-Regulates Pcsk1n Expression Thereby Regulating PC1/3 Dependent Proinsulin Processing

Background

Heterozygous paired box6 (Pax6) mutations lead to abnormal glucose metabolism in mice older than 6 months as well as in human beings. Our previous study found that Pax6 deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. As a protein cleaving enzyme, in addition to its expression, the activity of PC1/3 is closely related to its function. We therefore hypothesize that Pax6 mutation alters the activity of PC1/3, which affects proinsulin processing.

Methodology/Principal Findings

Using quantitative RT-PCR, western blot and enzyme assay, we found that PC1/3 C-terminal cleavage and its activity were compromised in Pax6 R266Stop mutant mice, and the expression of Pcsk1n, a potent inhibitor of PC1/3, was elevated by Pax6 deficiency in the mutant mice and MIN6 cells. We confirmed the effect of proSAAS, the protein encoded by Pcsk1n, on PC1/3 C-terminal cleavage and its activity by Pcsk1n RNAi in MIN6 cells. Furthermore, by luciferase-reporter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we revealed that Pax6 bound to Pcsk1n promoter and directly down-regulated its expression. Finally, by co-transfecting Pax6 siRNA with Pcsk1n siRNA, we showed that Pax6 knock-down inhibited proinsulin processing and that this effect could be rescued by proSAAS down-regulation. These findings confirm that Pax6 regulates proinsulin processing partially through proSAAS-mediated PC1/3 processing and activity.

Conclusions/Significance

Collectively, the above experiments demonstrate that Pax6 can directly down-regulate Pcsk1n expression, which negatively affects PC1/3 C-terminal cleavage and activity and subsequently participates in proinsulin processing. We identified proSAAS as a novel down-regulated target of Pax6 in the regulation of glucose metabolism. This study also provides a complete molecular mechanism for the Pax6 deficiency-caused diabetes.     

Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Association of telomere instability with senescence of porcine cells

Telomeres are essential for the maintenance of genomic stability, and telomere dysfunction leads to cellular senescence, carcinogenesis, aging, and age-related diseases in humans. Pigs have become increasingly important large animal models for preclinical tests and study of human diseases, and also may provide xeno-transplantation sources. Thus far, Southern blot analysis has been used to estimate average telomere lengths in pigs. Telomere quantitative fluorescence in situ hybridization (Q-FISH), however, can reveal status of individual telomeres in fewer cells, in addition to quantifying relative telomere lengths, and has been commonly used for study of telomere function of mouse and human cells. We attempted to investigate telomere characteristics of porcine cells using telomere Q-FISH method. The average telomere lengths in porcine cells measured by Q-FISH correlated with those of quantitative real-time PCR method (qPCR) or telomere restriction fragments (TRFs) by Southern blot analysis. Unexpectedly, we found that porcine cells exhibited high incidence of telomere doublets revealed by Q-FISH method, coincided with increased frequency of cellular senescence. Also, telomeres shortened during subculture of various porcine primary cell types. Interestingly, the high frequency of porcine telomere doublets and telomere loss was associated with telomere dysfunction-induced foci (TIFs). The incidence of TIFs, telomere doublets and telomere loss increased with telomere shortening and cellular senescence during subculture. Q-FISH method using telomere PNA probe is particularly useful for characterization of porcine telomeres. Porcine cells exhibit high frequency of telomere instability and are susceptible to telomere damage and replicative senescence.     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM642-1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM642-1290解旋酶与dsDNA的结合和解链特性．结果表明,BLM642-1290解旋酶与dsDNA的结合和解链和dsDNA3’末端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA3’末端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM642-1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3’末端ssDNA的dsDNA．推测BLM642-1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

东乡野生稻耐冷渐渗系的细胞学观察及SSR分析

以前期鉴定筛选的2个东乡野生稻强耐冷渐渗系(IL5243和IL5335)为试材,研究其减数分裂时期的染色体行为特征及外源基因的渗入分子证据。结果表明:(1)IL5243和IL5335中正常减数分裂的花粉母细胞分别达89.93%和90.22%,最终形成正常的成熟花粉粒,花粉离体萌发率分别为(83.03±2.82)%和(81.96±1.73)%,与受体亲本无显著性差异。(2)在减数分裂I中,2个耐冷渐渗系均观察到低频率异常染色体行为,如单价体、"8"字型二价体、多价体,以及后期I有少数花粉母细胞(3.95%～5.15%)存在落后染色体等,表明其染色体组之间发生了交换和重组;在粗线期,2个强耐冷渐渗系均观察到较高频率(IL5243和IL5335分别为27.0%和38.9%)的双核仁,而其双亲都是单核仁。(3)SSR标记和Structure分析进一步证实了栽培稻和野生稻染色体组间发生了交换重组,东乡野生稻部分DNA片段已渗入到强耐冷渐渗系中,这为水稻耐冷基因的挖掘与利用奠定了重要基础。     

贵州省城镇建设用地扩展的时空演变特征

选择贵州省的85个城镇为研究区,运用1973年1∶5万地形图,1986、1995、2000和2007年4期高分辨率遥感影像,选取城镇扩展动态度指数、城镇扩展贡献率指数和城镇扩展强度指数3个指标,构建城镇综合扩展程度指数模型,定量分析了1973—2007年4个时期贵州省城镇建设用地扩展的总体特征和时空分异特征。结果表明:从1973—2007年,贵州省城镇建设用地呈持续增长态势,城镇建设用地面积共增加17550.8915hm2,其中2000—2007年贵州省城镇建设用地扩展面积最大,动态度、贡献率和强度指数均最大;研究区建设用地扩展具有明显的空间分异特征,1973—1986年,城镇建设用地扩展主要集中在省会城市贵阳市,1986—1995年,地级城市所在城镇扩展面积和强度快速提高,1995—2000年,交通沿线城镇扩展速度加快,2000—2007年,贵州省城镇扩展强度和综合扩展程度不仅在贵阳市和地级城市较快,而且在区位和自然条件良好的县级城镇也迅速提高。     

杉木人工林土壤呼吸速率对水热条件变化的响应

利用LI-8100土壤碳通量观测仪,测定福建省建阳范桥林场杉木人工林水热条件天气变化下土壤呼吸的动态变化。结果表明,土壤呼吸速率与土壤温度变化趋势较为一致,但在不同天气下其峰值出现时间有所差异,正常天气情况下其峰值出现在13:00左右,当温度与降水均产生明显变化条件下,其峰值出现在11:00。与正常温度相比,在低温或高温状况下土壤呼吸的日变化幅度也发生改变,分别是37.9%和50.6%。观测期间,伴随大幅度降温降水,土壤呼吸速率呈急剧下降,在天气变化后的第5天达到最小值,随后随温度回升而增高,土壤呼吸从降低到恢复历经10d。统计分析表明,土壤呼吸与地表温度呈极显著正相关,与土壤体积含水量呈极显著负相关。     

探讨模拟冷空气降温过程对健康大鼠和高血压大鼠凝血功能的影响

目的:通过模拟冷空气温度变化过程给予健康大鼠和高血压大鼠冷刺激,以此探讨冷空气过程对机体凝血功能的影响。方法:收集张掖市2011年3月一次典型冷空气过程数据,利用气象环境模拟箱模拟其温度变化过程。将24只雄性健康大鼠和24只雄性高血压大鼠分别随机分成最低温组(Tmin组)、Tmin对照组、复温组(Tr组)和Tr对照组。将Tmin组和Tr组大鼠放入气候箱中暴露冷空气温度变化过程。在Tmin和Tr时点分别停止Tmin组大鼠和Tr组大鼠冷空气暴露,并采血以测定其凝血功能指标—凝血四项。结果:与对照组相比,健康大鼠和高血压大鼠活化部分凝血酶原时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)在降温后与对照组相比没有显著差异(P>0.05),但血浆纤维蛋白原(Fbg)含量在高血压大鼠和健康大鼠Tmin组明显高于其对照组(P<0.01)。温度恢复后,其在健康组含量仍然高于其对照组(P<0.05),而在高血压大鼠中没有差异(P>0.05)。同Fbg,反应Fbg的纤维蛋白原时间(Fbg-time)在健康大鼠Tmin和Tr组中短于对照组(P<0.01,),而在高血压大鼠中仅在Tmin组短于对照组(P<0.01)。高血压大鼠血中的Fbg含量和Fbgt明显高于和短于健康大鼠(P<0.01)。结论:①冷空气降温过程能增加机体血中Fbg含量,使凝血功能增强,可能增加心血管疾病危险性;②冷空气刺激对健康大鼠凝血功能影响强于高血压大鼠。     

利用B细胞培养和RT-PCR技术从我国HIV-1感染者中筛选膜蛋白特异性单克隆抗体的初步研究

本研究通过采集1型人类免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)感染者抗凝全血,分离出外周血单个核细胞,然后用磁珠分选纯化记忆性B细胞和体外活化记忆性B细胞,促使其分泌抗体,用ELISA法识别阳性B细胞克隆,并提取阳性B细胞的RNA,从中扩增抗体重链和轻链基因并克隆到表达载体中,再用携带重链基因的质粒和携带轻链基因的质粒共转染293T细胞,获得HIV-1特异性人单克隆抗体,进行抗体特性的鉴定。结果从1例HIV-1感染者的记忆性B细胞中筛选出了4株HIV-1包膜糖蛋白(Envelope glycoprotein,Env)特异性人单克隆抗体,其中2株具有较好的抗体依赖细胞介导的细胞毒作用活性,另有1株对HIV-1假病毒有较弱的中和活性。说明我们成功地引进了利用B细胞培养和RT-PCR技术从人体淋巴细胞中筛选特异性抗体基因的人单克隆抗体技术平台。用该技术可以成功获得HIV-1Env特异性单克隆抗体,为将来从能产生高滴度广谱中和抗体的感染者体内筛选广谱中和抗体打下了基础。